Quantikine™ ELISA Human CX3CL1/Fractalkine Immunoassay Catalog Number DCX310 For the quantitative determination of human Fractalkine concentrations in cell culture supernates, serum, plasma, saliva, urine, and human milk. This package insert must be read in its entirety before using this product. For research use only. Not for use in diagnostic procedures. TABLE OF CONTENTS SECTION PAGE INTRODUCTION .....................................................................................................................................................................1 PRINCIPLE OF THE ASSAY ...................................................................................................................................................2 LIMITATIONS OF THE PROCEDURE .................................................................................................................................2 TECHNICAL HINTS .................................................................................................................................................................2 MATERIALS PROVIDED & STORAGE CONDITIONS ...................................................................................................3 OTHER SUPPLIES REQUIRED .............................................................................................................................................3 PRECAUTIONS .........................................................................................................................................................................4 SAMPLE COLLECTION & STORAGE .................................................................................................................................4 SAMPLE PREPARATION........................................................................................................................................................4 REAGENT PREPARATION .....................................................................................................................................................5 ASSAY PROCEDURE .............................................................................................................................................................6 CALCULATION OF RESULTS ...............................................................................................................................................7 TYPICAL DATA .........................................................................................................................................................................7 PRECISION ................................................................................................................................................................................8 SENSITIVITY .............................................................................................................................................................................8 CALIBRATION ..........................................................................................................................................................................8 RECOVERY.................................................................................................................................................................................9 LINEARITY .................................................................................................................................................................................9 SAMPLE VALUES .................................................................................................................................................................. 10 SPECIFICITY ........................................................................................................................................................................... 11 REFERENCES ......................................................................................................................................................................... 12 PLATE LAYOUT ..................................................................................................................................................................... 13 Manufactured and Distributed by: USA R&D Systems, Inc. 614 McKinley Place NE, Minneapolis, MN 55413 TEL: 800 343 7475 612 379 2956 FAX: 612 656 4400 E-MAIL: [email protected] Distributed by: Europe | Middle East | Africa Bio-Techne Ltd. China Bio-Techne China Co., Ltd. 19 Barton Lane, Abingdon Science Park Unit 1901, Tower 3, Raffles City Changning Office, Abingdon OX14 3NB, UK 1193 Changning Road, Shanghai PRC 200051 TEL: +44 (0)1235 529449 TEL: +86 (21) 52380373 (400) 821-3475 FAX: +44 (0)1235 533420 FAX: +86 (21) 52371001 E-MAIL: [email protected] E-MAIL: [email protected] INTRODUCTION Fractalkine, also known as CX3CL1 or neurotactin, is the only member of the CX3C or delta chemokine subfamily (1-4). The name Fractalkine derives from the “fractal” nature of its discovery and placement within the chemokine family (4). Fractalkine is produced as a transmembrane protein, unlike all other chemokines except CXCL16 (1, 4). The 95-100 kDa type I transmembrane form of Fractalkine can be proteolytically processed to generate a secreted 60-80 kDa soluble form (4). Soluble Fractalkine has been identified in serum, urine, cerebrospinal, amniotic and synovial fluids (1, 5). The metalloproteinase ADAM10 cleaves Fractalkine constitutively, while TACE/ADAM17 does so inducibly (6, 7). Fractalkine contains an extracellular 76 amino acid (aa) chemokine domain that includes binding and chemotactic determinants, which is held away from the membrane by a 226 aa mucin-like stalk region (4, 8-10). Human Fractalkine shares 60% or 63% aa sequence identity within the entire extracellular domain, and 78% or 85% aa sequence identity within the chemokine domain only, with mouse or rat Fractalkine, respectively. Fractalkine is predominantly expressed by vascular endothelium and smooth muscle, neurons, dendritic cells, and the epithelial linings of the intestine, bronchi, renal proximal tubules, endometrium, fallopian tube, and bile duct (11-23). Most Fractalkine expression is induced by inflammatory cytokines such as TNF-α and IFN-γ, but expression on forebrain neurons as well as Leydig and Sertoli testicular cells is also constitutive (11-24). Expression by astrocytes, osteoblasts, stratum basal keratinocytes, and fibroblasts has also been reported (13, 17, 25, 26). CX3CR1, a 40 kDa 7-transmembrane non-glycosylated G-protein coupled receptor, is the only known endogenous receptor for Fractalkine (1-3). It is expressed by cytotoxic effector cells and cytokine producers, including type I helper and cytotoxic T cells, γδ T cells, CD16+ NK cells, monocytes and microglia (3, 13, 27, 28). In humans, three isoforms of the receptor that vary at the N-terminus differ in binding kinetics and chemotactic responses to the Fractalkine ligand (29, 30). Cytomegalovirus produces a protein called US28 that is also a receptor for human Fractalkine (8). The functions of Fractalkine are mainly dependent on its adhesion properties and chemoattractant activity and include some differences between soluble and transmembrane forms (1-3). Transmembrane Fractalkine, with cooperation from platelet P-Selectin under arterial high shear, mediates firm adhesion to circulating CX3CR1-expressing cells (6, 12, 31). Cleavage of the membrane-bound Fractalkine may then free adhering leukocytes to allow diapedesis (6, 10, 12, 32). Both soluble and transmembrane forms are chemoattractants that bring cytotoxic effector and cytokine producing cells to areas of inflammation (1-3, 23, 26, 27). Fractalkine, especially in the soluble form, enhances the cytotoxic response of CX3CR1-expressing NK cells, which may be responsible for its antitumor activity (33, 34). Fractalkine is thought to mediate interactions between neurons and microglia in the brain, and to protect the brain against microglial neurotoxicity during neuroinflammation (35, 36). Production of Fractalkine by the endometrium is postulated to promote trophoblast migration for implantation, and its abundance in endometrium and placenta implies presence of Fractalkine in the plasma during pregnancy (37). While Fractalkine is important in resolving normal inflammatory processes such as wound healing, it may also contribute to the pathogenesis of some conditions (3, 38). For example, both CX3CR1 and soluble Fractalkine are often over-expressed in coronary artery disease and may contribute to arterial plaque destabilization (39). Fractalkine may also be upregulated in systemic lupus erythematosis and in rheumatoid arthritis synovial fluid (1, 40). The Quantikine™ Human CX3CL1/Fractalkine Immunoassay is a 4.5 hour solid phase ELISA designed to measure human Fractalkine in cell culture supernates, serum, plasma, saliva, urine, and human milk. It contains NS0-expressed recombinant human Fractalkine and has been shown to accurately quantitate the recombinant factor. Results obtained using natural Fractalkine showed linear curves that were parallel to the standard curves obtained using the Quantikine kit standards. These results indicate that this kit can be used to determine relative mass values for natural human Fractalkine. www.RnDSystems.com 1 PRINCIPLE OF THE ASSAY This assay employs the quantitative sandwich enzyme immunoassay technique. A monoclonal antibody specific for human Fractalkine has been pre-coated onto a microplate. Standards